WO2018016483A1 - Method for producing mogrol or mogrol glycoside - Google Patents

Abstract

A novel method for producing a mogrol glycoside and mogrol has been required. The present invention provides a method for producing a mogrol glycoside and/or mogrol, said method comprising a step for cleaving at least one glucoside bond in a mogrol glycoside.

Description

Method for producing mogrol or mogrol glycoside

The present invention relates to a method for producing mogrol or mogrol glycoside.

Lacanka (Siraitia grosvenorii) is a cucurbitaceae plant originating in Guangxi, China. Lacanca fruit exhibits strong sweetness, and its extract is used as a natural sweetener. In addition, dried lakanka fruit is also used as a herbal medicine.
Lacanka fruit is known to contain mogrol glycoside as a sweetening ingredient. A mogrol glycoside is a glycoside in which glucose is bound to mogrol, which is an aglycone. Mogrol glycosides are classified into various mogrol glycosides depending on the binding position and number of glucose. Mogrol glycosides contained in the fruits of Lacanca are mogroside V, mogroside IV, siamenoside I and 11-oxomogroside. In addition, the Moguroru glycosides, mogrosides I, mogrosides IVA, mogroside III, mogroside IIIA _1, mogroside IIIA _2, mogroside IIIE, mogroside IIA, mogroside IIA _1, mogroside IIA _2, mogroside IIB, mogroside IIE, mogroside IA ₁ And mogroside IE ₁ are known.
It is known that mogrol, which is an aglycon, has an anticancer action as a physiologically active action (Non-patent Document 1).

There are several known methods for preparing mogrol from mogrol glycosides, for example, a method of acid hydrolysis by heating mogrol glycosides in 0.5N HCl at 95-100 ° C. for 10 hours is disclosed. (Non-Patent Document 1).

In addition, a method of hydrolyzing mogrol glycoside with an enzyme is known (Patent Document 1). In this method, specifically, pectinase derived from Aspergillus niger is added and reacted at 50 ° C. for 48 hours. However, it is not clear about the enzyme protein responsible for this activity and the gene encoding it.

It is disclosed that yeast (Saccharomyces cerevisiae) has an activity of converting mogroside V to mogroside IIIE, and that the gene of the enzyme responsible for the activity is EXG1 (GH5 family, β-1,3 glucanase) ( Non-patent document 2).

WO2013 / 07577

Under such circumstances, a new production method of mogrol and / or mogrol glycoside has been demanded.

As a result of intensive studies to solve the above problems, the present inventors have found that AOBGL11p, which is a glycoside hydrolase derived from Aspergillus oryzae, has an activity of hydrolyzing mogroside V to produce mogrol and mogrol glycoside. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1)
A step of reacting a protein selected from the group consisting of the following (a) to (c) with a mogrol glycoside to hydrolyze at least one glucoside bond of the mogrol glycoside, and / or A method for producing mogrol glycoside.
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted and / or added, and hydrolyzes at least one glucoside bond of the mogrol glycoside. A protein having the activity of:
(C) a protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside (2) )
In the production method of (1), the mogrol glycoside to be reacted with the protein is mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, and mogroside includes mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , mogroside The above-mentioned (1), which is at least one selected from the group consisting of IIIA ₂ , mogroside IIIE, mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE ₁ Method.
(3)
The method according to (1) or (2), wherein the mogrol glycoside is at least one selected from the group consisting of mogroside V, mogroside IIIE, and siamenoside I.
(4)
The method according to any one of (1) to (3), wherein the mogrol glycoside is mogrol V.
(5)
The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a glucoside bond between glucose added at the 3-position of mogrol and mogrol which is an aglycone, and at the 24-position of mogrol. Β-1,6-glucoside bond of added branched trisaccharide, β-1,6-glucoside bond of gentiobiose added at position 24 of mogrol, β-1,2-glucoside of sophorose added at position 24 of mogrol The method according to any one of (1) to (4) above, which is any one of a bond and / or a glucoside bond between glucose added to position 24 of mogrol and mogrol which is an aglycone.

(6)
An enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to (e) is introduced into a host cell is contacted with the mogrol glycoside, and the mogrol A method for producing mogrol and / or mogrol glycoside, comprising hydrolyzing at least one glucoside bond of the glycoside.
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added, and at least one glucoside bond of the mogrol glycoside is hydrolyzed. A polynucleotide encoding a protein having an activity of degrading;
(D) encoding a protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. A polynucleotide to:
(E) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and at least one of the mogrol glycosides A polynucleotide encoding a protein having an activity of hydrolyzing a glucoside bond (7)
The method according to (6) above, wherein the polynucleotide is inserted into an expression vector.
(8)
The method according to (6) or (7), wherein the transformed cell is a transformed gonococcus, a transformed yeast, a transformed bacterium, or a transformed plant.
(9)
The mogrol glycoside to be brought into contact with the enzyme agent is mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, mogroside includes mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside IIIE, mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE ₁ are any one of the above (6) to (8) The method described.
(10)
The method according to any one of (6) to (9), wherein the mogrol glycoside is at least one selected from the group consisting of mogroside V, mogroside IIIE, and siamenoside I.
(11)
The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a glucoside bond between glucose added at the 3-position of mogrol and mogrol which is an aglycone, and at the 24-position of mogrol. Β-1,6-glucoside bond of added branched trisaccharide, β-1,6-glucoside bond of gentiobiose added at position 24 of mogrol, β-1,2-glucoside of sophorose added at position 24 of mogrol The method according to any one of (6) to (10) above, which is any one of a bond and / or a glucoside bond between glucose added to position 24 of mogrol and mogrol which is an aglycone.
(12)
A method for producing mogrol and / or mogrol glycoside, comprising culturing a non-human transformant into which a polynucleotide selected from the group consisting of the following (a) to (e) is introduced.
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added, and at least one glucoside bond of the mogrol glycoside is hydrolyzed. A polynucleotide encoding a protein having an activity of degrading;
(D) encoding a protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. A polynucleotide to:
(E) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and at least one of the mogrol glycosides Polynucleotide encoding protein having activity of hydrolyzing glucoside bond (13)
The method according to (10) above, wherein the polynucleotide is inserted into an expression vector.
(14)
The method according to (12) or (13), wherein the transformant is a transformed gonococcus, a transformed yeast, a transformed bacterium, or a transformed plant.
(15)
The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a β-1,6-glucoside bond of a branched trisaccharide added at the 24-position of mogrol, and / or mogrol The method according to any one of (12) to (14) above, which is any one of β-1,2-glucoside bonds of a branched trisaccharide added at position 24.

The method of the present invention provides a new method for producing mogrol and mogrol glycoside.

Moreover, various mogrol glycosides can be produced by selecting reaction conditions. In one embodiment of the present invention, the production amount of mogrol and / or mogrol glycoside can be increased by selecting reaction conditions such as enzyme concentration. Further, according to one embodiment of the present invention, it is possible to selectively increase the production amount of mogrol tetrasaccharide glycosides such as siamenoside I, mogrol trisaccharide glycosides such as mogroside IIIE, and mogrol disaccharide glycoside. it can.

CDNA sequence and amino acid sequence of AOBGL11. CDNA sequence and amino acid sequence of AOBGL11. A: Optimum temperature of AOBGL11p using pNP-β-Glc as a substrate. B: Optimum pH of AOBGL11p using pNP-β-Glc as a substrate. C: Thermal stability of AOBGL11p using pNP-β-Glc as a substrate. D: pH stability of AOBGL11p using pNP-β-Glc as a substrate. Comparison of AOBGL11 genomic and cDNA sequences. Comparison of AOBGL11 genomic and cDNA sequences. Reaction of BGL11-1 crude enzyme solution with mogroside V. A: No substrate, no dilution of BGL11-1 crude enzyme solution. B: Mogroside V was added as a substrate, and the dilution ratio of the BGL11-1 crude enzyme solution was 100 times. C: Mogroside V was added as a substrate, and the dilution ratio of the BGL11-1 crude enzyme solution was 10 times. D: Mogroside V was added as a substrate, and BGL11-1 crude enzyme solution was not diluted. Reaction of C-1 crude enzyme with mogroside V. A: No substrate, B: Mogroside V added as substrate. All C-1 crude enzyme solutions were used without dilution.

Hereinafter, the present invention will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone. The present invention can be implemented in various forms without departing from the gist thereof.
It should be noted that all documents cited in the present specification, as well as published publications, patent gazettes, and other patent documents are incorporated herein by reference. In addition, this specification includes the contents described in the specification and drawings of the Japanese patent application (Japanese Patent Application No. 2016-1416885), which is the basis of the priority claim of the present application filed on July 19, 2016.

Hereinafter, the mogrol glycoside as a substrate is sometimes referred to as “substrate mogrol glycoside”, and the mogrol glycoside as a product is sometimes referred to as “produced mogrol glycoside”. In addition, both may be collectively referred to as “mogrol glycosides”.
“AOBGL11p” is β-glucosidase derived from Aspergillus oryzae, the cDNA sequence is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2, and the genomic DNA sequence is shown in SEQ ID NO: 3.

1. The present invention relates to a protein selected from the group consisting of the following (a) to (c) (hereinafter referred to as “protein of the present invention”), and a mogrol glycoside. There is provided a method for producing mogrol and / or mogrol glycoside, which comprises reacting and hydrolyzing at least one glucoside bond. In one embodiment, the present invention provides a method for producing mogrol comprising the step of cleaving all glucoside bonds of the mogrol glycoside. In another embodiment, the present invention provides specific linkages of mogrol glycosides, such as the β-1,6-glucoside bond of gentiobiose added at position 3 of mogrol and / or the branching added at position 24 of mogrol. Provided is a method for producing mogrol and / or mogrol glycoside, which comprises a step of preferentially cleaving a β-1,6-glucoside bond of a trisaccharide. In this case, in addition to cleaving the β-1,6-glucoside bond, a step of cleaving the β-1,2-glucoside bond of the branched trisaccharide may be included.

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted and / or added, and hydrolyzes at least one glucoside bond of the mogrol glycoside. A protein having the activity of:
(C) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of mogrol glycoside

The protein described in the above (b) or (c) is typically a variant of a protein consisting of the amino acid sequence of SEQ ID NO: 2, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor Laboratory Press 2012 ”,“ Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 ”,“ Nuc. Acids. Res., 10, 6487 (1982) ”,” Proc. Natl. Acad .Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”and the like, which can be obtained artificially using the site-directed mutagenesis method described in the above.

“The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted and / or added, and an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. As the “protein having”, in the amino acid sequence of SEQ ID NO: 2, for example, 1 to 83, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55 1 to 50, 1 to 49, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to 41 1 to 40 1 to 39 1 to 38 1 to 37 1 to 36 1 to 35 1 to 34 1 to 33 1 to 32 1 to 31 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 4, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1-6 Consisting of amino acid sequences in which ˜5, 1-4, 1-3, 1-2, or 1 amino acid residues are deleted, substituted, inserted and / or added, and of mogrol glycosides Examples include proteins having an activity of hydrolyzing at least one glucoside bond. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better.

Such a protein includes the amino acid sequence of SEQ ID NO: 2, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99. Examples thereof include a protein having an amino acid sequence having a sequence identity of 8% or more, or 99.9% or more and having an activity of hydrolyzing at least one glucoside bond of a mogrol glycoside. In general, the larger the sequence identity value, the better.

Here, “at least one glucoside bond of mogrol glycoside” means a glucoside bond between mogrol, which is an aglycon, and a side chain in mogrol glycoside, which is a glycoside in which sugar is bound to mogrol, and Means a glucoside bond in the side chain. In addition, the “activity of hydrolyzing at least one glucoside bond of a mogrol glycoside” means that the mogrol glycoside is a glycoside in which sugar is bound to mogrol, between mogrol that is an aglycone and the side chain. It means an activity of cleaving (hydrolyzing) at least one of a glucoside bond and a glucoside bond in a side chain. Examples of glucoside linkages in the side chain include β-1,6-glucoside linkage of gentiobiose added at position 3 of mogrol, β-1,6-glucoside linkage of branched trisaccharide at position 24 of mogrol, position 24 of mogrol Β-1,2-glucoside bond of branched trisaccharide, β-1,6-glucoside bond of gentiobiose added at position 24 of mogrol, and / or β-1,2- of sophorose added at position 24 of mogrol A glucoside bond is mentioned. In certain embodiments, all glucoside bonds are hydrolyzed to yield mogrol from the mogrol glycoside. In another embodiment, the β-1,6-glucoside bond of gentiobiose added at position 3 of mogrol and / or the β-1,6-glucoside bond of the branched trisaccharide at position 24 of mogrol is preferentially hydrolyzed. Is done. In this way, mogrol glycosides in which only some of the sugars are cleaved are produced.

The activity of hydrolyzing at least one glucosidic bond of mogrol glycosides can be achieved with the protein of the present invention, for example, mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , Reacting at least one mogrol glycoside selected from the group consisting of mogroside IIIA ₂ , mogroside IIIE, mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE ₁ The obtained reaction product (mogrol and / or mogrol glycoside) is purified, and the purified product is purified by a known technique such as liquid chromatography (LC). It can be confirmed by analysis.

“In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acid residues are deleted, substituted, inserted and / or added” means any position in the same sequence and 1 to 83 amino acid sequences Means that there are deletion, substitution, insertion and / or addition of 1 to 83 amino acid residues, and two or more of deletion, substitution, insertion and addition may occur simultaneously.

Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
Group C: asparagine, glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E: proline, 3-hydroxyproline, 4-hydroxyproline;
Group F: serine, threonine, homoserine;
Group G: phenylalanine, tyrosine.

The protein of the present invention can be obtained by, for example, expressing a polynucleotide encoding the same (see “polynucleotide of the present invention” described later) in an appropriate host cell. The Fmoc method (fluorenylmethyl) It can also be produced by chemical synthesis methods such as the oxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Also, AAPPPec LLC, Perkin Elmer Inc. Manufactured by Protein Technologies Inc. Chemical synthesis can also be carried out using a peptide synthesizer such as manufactured by PerSeptive Biosystems, Applied Biosystems, or SHIMADZU CORPORATION.

In the present invention, the “mogrol glycoside” is a glycoside in which a sugar is bound to mogrol. Examples of mogrol and mogrol glycoside are represented by the following general formula (I). The 3rd position of mogrol and the 24th position of mogrol are shown in the formula. In the present invention, the “branched trisaccharide” refers to those constituting a trisaccharide among those added to the R ₂ moiety in the following general formula (I).

Examples of mogrol glycosides are mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside IIIE, mogroside IIA, mogroside IIA ₁ , mogroside Examples include, but are not limited to, IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE _{1 and the} like.

In one embodiment of the present invention, the enzyme of the present invention uses a protein consisting of the amino acid sequence of SEQ ID NO: 2 and / or a protein consisting of a variant thereof. In another embodiment, mogrol glycosides can be utilized as a substrate. Here, the example of a mogrol glycoside is as above-mentioned. In one embodiment of the present invention, using the enzyme of the present invention, the reaction conditions can be adjusted by adjusting the amount of enzyme added to the reaction, adding an organic solvent to the reaction solution, or adjusting the reaction temperature. By adjusting the mogroside V, which is the most abundant mogrol glycoside in Lacanca fruit, as a substrate, it is rare such as mogroside IIIE and mogroside II (mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE). It is also possible to produce mogrol glycosides.

In the present invention, the enzyme amount, substrate / enzyme ratio, temperature, pH, presence / absence of a solvent, reaction time, etc. of the reaction using mogrol glycoside as a substrate can be adjusted as appropriate by those skilled in the art. It is possible to control the degree of degradation to what extent the sugar bound to the saccharide is cleaved.

The method for producing mogrol and / or mogrol glycoside according to the present invention hydrolyzes at least one glucoside bond of mogrol glycoside.

In the method for producing mogrol and / or mogrol glycoside of the present invention, the starting mogrol glycoside is obtained from Siraitia grosvenorii as an appropriate solvent (aqueous solvent such as water or organic such as alcohol, ether and acetone). Solvent) extraction, ethyl acetate and other organic solvents: water gradient, high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (ultra (liquid) liquid chromatography: UPLC), etc. It can obtain by extracting and refine | purifying by the method of this. Alternatively, the starting mogrol glycoside may be commercially available. Mogrol glycosides as starting materials of the present invention include mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside IIIE, mogroside IIA, Mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE ₁ and various mogrol glycosides by cleavage of β-1,6-glucoside bond or β-1,2-glucoside bond Such as siamenoside I, mogroside IIIE, and mogrol disaccharide glycoside.

The method for producing mogrol and / or mogrol glycoside according to the present invention includes a step of hydrolyzing at least one glucoside bond of mogrol glycoside by reacting the protein of the present invention with mogrol glycoside. The method of the present invention may further comprise a step of purifying the mogrol and / or mogrol glycoside of the present invention produced in the above step.
The mogrol and / or mogrol glycoside of the present invention is extracted with an appropriate solvent (aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone), ethyl acetate and other organic solvents: water gradient, high speed It can be purified by known methods such as liquid chromatography (HPLC), ultra high performance liquid chromatography (Ultra (High) Performance Chromatography: UPLC), and the like.

The method for producing a mogrol glycoside and / or mogrol according to the present invention can be performed under a condition in which an organic solvent is added to a reaction solution containing a substrate. The organic solvent can be in the range of 1% to 20% with respect to the total amount of the reaction solution, preferably 5% to 15%, 6 to 12%, more preferably 8%. The organic solvent may be generally available, preferably mixed with water in an arbitrary ratio, and acetonitrile or the like can be used. The organic solvent may be added in advance to the reaction solution, or may be added in the middle of the reaction.

In the present specification, “polynucleotide” means DNA or RNA.

Examples of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 include a polynucleotide consisting of the base sequence of SEQ ID NO: 1.

“The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted and / or added, and an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. Examples of the “protein having” include those described above.

“A protein having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of a mogrol glycoside” Can be mentioned.

In the present specification, “a polynucleotide that hybridizes under highly stringent conditions”, for example, encodes a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. A polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using as a probe all or part of a polynucleotide comprising a base sequence complementary to the base sequence to be detected. Examples of the hybridization method include “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor, Laboratory Press 2012”, “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997”. Can be used.
In the present specification, “high stringent conditions” means, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C., or 0.2 × SSC, 0.1% SDS, The conditions are 60 ° C., 0.2 × SSC, 0.1% SDS, 62 ° C., or 0.2 × SSC, 0.1% SDS, 65 ° C., but are not limited thereto. Under these conditions, it can be expected that DNA having high sequence identity can be efficiently obtained as the temperature is increased. However, factors affecting the stringency of hybridization include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can select these factors as appropriate. By doing so, it is possible to achieve the same stringency.

In addition, when using a commercially available kit for hybridization, Alkphos Direct Labeling and Detection System (GE Healthcare) can be used, for example. In this case, according to the protocol attached to the kit, after overnight incubation with the labeled probe, the membrane was subjected to a primary wash containing 0.1% (w / v) SDS at 55-60 ° C. After washing with a buffer, the hybridized DNA can be detected. Alternatively, when preparing a probe based on a base sequence complementary to the base sequence of SEQ ID NO: 1 or a base sequence complementary to the base sequence encoding the amino acid sequence of SEQ ID NO: 2, commercially available When the reagent is labeled with digoxigenin (DIG) using a reagent (for example, PCR labeling mix (Roche Diagnostics)), hybridization is performed using a DIG nucleic acid detection kit (Roche Diagnostics). Can be detected.

In addition to the above, the hybridizable polynucleotide encodes the DNA of the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, when calculated by the BLAST homology search software using the default parameters. 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% Above, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, Mention may be made of DNA having a sequence identity of 99.7% or more, 99.8% or more, or 99.9% or more.

The sequence identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Basic Local Alignment Search Tool) by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). When using BLAST, the default parameters of each program are used.

The above-described polynucleotide of the present invention can be obtained by a known genetic engineering technique or a known synthesis technique.

The polynucleotide of the present invention may further include a polynucleotide consisting of a base sequence encoding a secretory signal peptide. Preferably, a polynucleotide consisting of a base sequence encoding a secretory signal peptide is included at the 5 'end of the polynucleotide of the present invention. The polynucleotide from the secretory signal peptide and the base sequence encoding it is the same as described above.

The polynucleotide of the present invention is preferably introduced into a host while being inserted into an appropriate expression vector.

Suitable expression vectors are usually
(I) a promoter capable of transcription in a host cell;
(Ii) a polynucleotide of the present invention linked to the promoter; and (iii) an expression cassette comprising, as a component, a signal that functions in a host cell for transcription termination and polyadenylation of an RNA molecule. .

The method for producing the expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like.

The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good.

The expression vector of the present invention contains an expression control region (for example, a promoter, a terminator and / or a replication origin) depending on the type of host to be introduced. Conventional promoters (eg, trc promoter, tac promoter, lac promoter, etc.) are used as promoters for bacterial expression vectors, and examples of yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. Examples of the promoter for filamentous fungi include amylase and trpC. Examples of promoters for expressing a target gene in plant cells include the cauliflower mosaic virus 35S RNA promoter, the rd29A gene promoter, the rbcS promoter, and the enhancer sequence of the cauliflower mosaic virus 35S RNA promoter derived from Agrobacterium. And the mac-1 promoter added to the 5 'side of the mannopine synthase promoter sequence. Examples of animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.). Examples of promoters that are inducibly activated by external stimuli include a mouse mammary tumor virus (MMTV) promoter, a tetracycline responsive promoter, a metallothionein promoter, a heat shock protein promoter, and the like.

The expression vector preferably contains at least one selectable marker. Such markers include auxotrophic markers (ura5, niaD), drug resistance markers (hygromycin, zeocin), geneticin resistance gene (G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, vol. 81, p. 337, セ ル 1984), cerulenin resistance gene (fas2m, PDR4) (respectively, Koshiwa 淳 嗣 et al., Biochemistry, vol. 64, p. , Gene, vol. 101, p. 149, 1991) can be used.

The production method (production method) of the transformant of the present invention is not particularly limited, and examples thereof include a method of transforming by introducing an expression vector containing the polynucleotide of the present invention into a host. As cells or organisms to be transformed, various conventionally known cells or organisms can be suitably used. Examples of cells to be transformed include bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), filamentous fungi (gonococci Aspergillus oryzae, Aspergillus sojae), plant cells, and humans. Excluding animal cells and the like. Appropriate culture media and conditions for the above-described host cells are well known in the art. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms exemplified in the host cell, animals other than plants or animals. The transformant is preferably a filamentous fungus, a yeast or a plant.
As a host used in transformation, a host that produces any of mogrol glycosides can also be used. Like Lacanca, not only plants that originally produce at least one mogrol glycoside, but also cells or organisms that do not naturally produce mogrol glycoside have been introduced with a gene necessary for the production of at least one mogrolol glycoside. Can be used as a host. Examples of the “gene necessary for the production of mogrol glycoside” include genes having mogrol glycoside synthesis activity described in WO2016 / 050890 and the like.

As a host cell transformation method, a commonly known method can be used. For example, an electroporation method (Mackenxie, D. A. et al., Appl. Environ. Microbiol., Vol. 66, p. 4655-4661, デ リ バ リ ー 2000), particle delivery method (Japanese Patent Laid-Open No. 2005-287403, “lipid producing bacteria”) ), Spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978), lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983), Methods inyeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual), but is not limited thereto. In addition, for gene transfer into plants or tissues or cells derived from plants, for example, Agrobacterium method (PlantMolecular Biology Manual, Gelvin, vinS. B. et al., Academic Press Publishers), particle gun method PEG method, electroporation method and the like can be appropriately selected and used.

When the transformant is yeast or koji mold, the yeast or koji mold transformed with the polynucleotide of the present invention expresses more of the protein of the present invention than the wild type. For this reason, the expressed protein of the present invention reacts with a mogrol glycoside produced in yeast or koji mold, and mogrol and / or mogrol glycoside is preferably cultured in the yeast or koji mold cells or in the culture solution. Produced in liquid.

When the transformant is a plant, the plant to be transformed in the present invention is the whole plant, plant organ (eg, leaf, petal, stem, root, seed, etc.), plant tissue (eg, epidermis, phloem) , Soft tissue, xylem, vascular bundle, palisade tissue, spongy tissue, etc.) or plant culture cells, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, etc. Also means. The plant used for the transformation may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class. Whether or not the polynucleotide of the present invention has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. Once a transformed plant in which the polynucleotide of the present invention has been integrated into the genome is obtained, offspring can be obtained by sexual or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, and the like can be obtained from the plant or its progeny, or clones thereof, and the plant can be mass-produced based on them. it can. A plant transformed with the polynucleotide of the present invention (hereinafter, “plant of the present invention”) contains more of the protein of the present invention than its wild type. For this reason, the protein of this invention reacts with the mogrol glycoside produced | generated with the plant of this invention, and mogrol produces | generates in a plant. In addition, when the environment in the plant body is not optimal for the hydrolysis reaction, the hydrolysis reaction of the glucoside bond of the mogrol glycoside is suppressed, and the mogrol glycoside or glucoside bond is maintained without breaking the glucoside bond. Mogrol glycosides that are cleaved only in part are produced.

Thus, as another embodiment, the present invention provides a method for producing mogrol and / or mogrol glycoside, which comprises culturing a non-human transformant introduced with the polynucleotide of the present invention.

The transformant of some aspects of the present invention or its culture solution has a higher content of the mogrol and / or mogrol glycoside of the present invention than its wild type, and the extract or culture solution contains The inventive mogrols and / or mogrol glycosides are included in high concentrations. The extract of the transformant of the present invention can be obtained by crushing the transformant using glass beads, a homogenizer, a sonicator or the like, centrifuging the crushed material, and collecting the supernatant. it can. When the mogrol and / or mogrol glycoside of the present invention is accumulated in the culture solution, the transformant and the culture supernatant are separated by a usual method (for example, centrifugation, filtration, etc.) after completion of the culture. By doing so, a culture supernatant containing the mogrol and / or mogrol glycoside of the present invention can be obtained.

The thus obtained extract or culture supernatant may be further subjected to a purification step. The purification of mogrol and / or mogrol glycoside of the present invention can be carried out according to ordinary separation and purification methods. The specific method is the same as described above.

Method for producing mogrol glycoside and / or mogrol of the present invention using an enzyme agent derived from a non-human transformed cell The protein of the present invention is expressed by expressing the protein of the present invention in a host cell and disrupting the cell. Obtainable. By allowing the protein of the present invention to act, the mogrol glycoside and / or mogrol of the present invention can also be produced.
Specifically, mogrol can be produced by contacting an enzyme agent derived from the transformed cell of the present invention with a mogrol glycoside having at least one glucoside bond. It has been confirmed in the Examples that the protein of the present invention exhibits the same activity when expressed in yeast as in the case of expression in Aspergillus. "Enzyme agent derived from transformed cells" is not limited as long as it is prepared using transformed cells and contains the protein of the present invention. For example, transformed cells themselves, pulverized products of transformed cells themselves, The culture supernatant of the transformed cell itself, and purified products thereof. Accordingly, the present invention provides an enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to (e) is introduced into a host cell, and at least one glucoside bond: A method for producing mogrol and / or mogrol glycoside comprising the step of hydrolyzing at least one glucoside bond by contacting with the mogrol glycoside having the formula:
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added, and hydrolyzes at least one glucoside bond of the mogrol glycoside. A polynucleotide encoding a protein having the activity of:
(D) a protein protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of mogrol glycoside A polynucleotide to:
(E) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 and at least one glucoside of a mogrol glycoside Polynucleotide encoding a protein having activity of hydrolyzing a bond

The polynucleotide selected from the group consisting of (a) to (e) above is the polynucleotide of the present invention and is the same as described above.

“Contact” means that an enzyme agent derived from the transformed cell of the present invention and a mogrol glycoside having at least one glucoside bond are present in the same reaction system or culture system. Addition of a mogrol glycoside having at least one glucoside bond to a container containing an enzyme agent derived from the transformed cell of the invention, an enzyme agent derived from the transformed cell of the present invention and mogrol having at least one glucoside bond Mixing with a glycoside and adding an enzyme agent derived from the transformed cell of the present invention to a container containing a mogrol glycoside having at least one glucoside bond are included.

The “mogrol glycoside”, “mogrol glycoside having at least one glucoside bond”, and “activity for hydrolyzing at least one glucoside bond” are the same as described above.

Other general molecular biology methods include “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor Laboratory Press 2012”, “Methods in Yeast Genetics, A laboratory manual (Cold Spring Hual "Laboratory (Press, Cold, Spring, Harbor, NY)".

The thus obtained mogrol and / or mogrol glycoside of the present invention can be produced, for example, according to a conventional method such as production of foods, sweeteners, fragrances, pharmaceuticals, industrial raw materials (raw materials such as cosmetics and soaps). Can be used for applications.

Examples of foods include nutritional supplements, health foods, functional foods, infant foods, and elderly foods. In the present specification, food is a general term for solids, fluids, liquids, and mixtures thereof that can be consumed.

It should be noted that all documents cited in the present specification, as well as published publications, patent gazettes, and other patent documents are incorporated herein by reference.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

[Example 1]
Search for β-glucosidase gene of Aspergillus by searching for β-glucosidase homologue from Aspergillus genome data (PRJNA28175), AO090700024044 (CDS sequence: SEQ ID NO: 1, deduced amino acid sequence: SEQ ID NO: 2, The ORF sequence: SEQ ID NO: 3, and the genomic DNA sequence: SEQ ID NO: 4) were found. We decided to clone this as AOBGL11. The cDNA sequence and amino acid sequence of AOBGL11 are shown in FIG.

Cloning of AOBGL11 genomic DNA In order to clone AOBGL11, the following primers were designed.
AOBGL11-F:
5′-ATGCCTCGTCTAGACCGTCGAGAA-3 ′ (SEQ ID NO: 4)
AOBGL11-R:
5'-TCACAGACCCAACCAGTAGGCGA-3 '(SEQ ID NO: 5)

Conidia of Aspergillus oryzae var. Brunneus (IFO30102) were added to a liquid medium (1 g, glucose 20 g, bactotryptone 1 g, yeast extract 5 g, NaNO ₃ 1 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ · 7H ₂ O 0 0.5 g, FeSO ₄ · 7H ₂ O 0.01 g) was inoculated into 10 ml, and cultured at 30 ° C. for 1 day. Bacteria were collected by filtration, ground in liquid nitrogen, and then genomic DNA was prepared using DNeasy Plant Mini Kit (QIAGEN).
PCR was performed with KOD-plus (Toyobo) using genomic DNA as a template and primers AOBGL11-F and AOBGL11-R. The obtained DNA fragment of about 2.57 kbp was cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) to obtain plasmid pCR-AOBGL11g.

[Example 2]
Production of AOBGL11p by Aspergillus Construction of a vector for expressing Aspergillus oryzae DNA fragment obtained by digestion of Aspergillus oryzae vector pUNA (Liquor Research Institute) with restriction enzyme SmaI and plasmid pCR-AOBGL11g with restriction enzyme EcoRI and blunting ends The DNA fragment of about 2.57 kbp obtained by blunting with Kit (Takara Bio) was ligated to obtain 11 g of plasmid pUNA-AOBGL.

Transformation of koji molds Koji molds were transformed as follows.
As a host, Aspergillus oryzae niaD300 strain (Liquor Research Institute) was used. Host strains were inoculated on PDA plates and cultured at 30 ° C. for approximately 1 week. To obtain a conidial suspension, 0.1% tween 80, 0.8% NaCl was added to suspend the conidia. After filtration through Miracloth, conidia were collected by centrifugation, further washed with 0.1% tween 80, 0.8% NaCl, and suspended in sterile water.
Conidia were added to CD plates (6 g NaNO ₃ , 0.52 g KCl _2, 1.52 g KH ₂ PO ₄ , ₂ g glucose, 1 ml MgSO ₄ , trace element solution (1 g FeSO ₄ · 7H ₂ O, 1 g ZnSO). 4.8 g of ₄ · 7H ₂ O, 0.4 g of CuSO ₄ · 5H ₂ O, 0.1 g of NaB ₄ O ₇ · 10H ₂ O, (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O 0.05 g) 1 ml, Agar 20 g (pH 6.5) was applied, and DNA was introduced by a particle delivery method. Using PDS-1000 / He (Bio-Rad), particle: tungsten M-10, rupture disk: 1100 psi, distance: 3 cm. As a transformant, one that grows on a CD plate was selected. The transformant obtained by transforming with plasmid pUNA-AOBGL11g was designated as BGL11-1 strain, and the transformant obtained by transformation with control vector pUNA was designated as C-1 strain.

Expression of AOBGL11p by Aspergillus BGL11-1 or C-1 strain was inoculated on a CD plate and cultured at 30 ° C. for 7 days to form conidia. To obtain a conidial suspension, 0.1% tween 80, 0.8% NaCl was added to suspend the conidia. After filtration through Miracloth (registered trademark), conidia are collected by centrifugation, further washed with 0.1% tween 80, 0.8% NaCl, suspended in sterilized water to obtain a conidia suspension. A liquid medium for enzyme production (100 g of maltose, 1 g of bactotryptone, 5 g of yeast extract, 1 g of NaNO ₃ , 0.5 g of K ₂ HPO _4, 0.5 g of MgSO ₄ .7H ₂ O, FeSO ₄ 7H ₂ O (0.01 g) was inoculated and cultured with shaking at 30 ° C. for 2 days. The cells were collected by filtration through Miracloth (registered trademark). About 4 g of the obtained wet cells were frozen with liquid nitrogen and ground in a mortar. The ground cells were suspended in 50 mM sodium phosphate buffer (pH 7.0), mixed well, and then centrifuged. The obtained supernatant is concentrated by ultrafiltration with Amicon (registered trademark) Ultra-15 50k (Merck) and replaced with 50 mM sodium phosphate buffer pH 7.0 (buffer A) containing 0.1% CHAPS. As a result, about 1 ml of a crude enzyme solution was obtained.

Measurement of protein concentration The protein concentration of the crude enzyme solution was quantified using a protein assay CBB solution (concentrated 5 times) (Nacalai Tesque). As a result, the BGL11-1 crude enzyme solution was 6.46 mg / ml and the C-1 crude enzyme solution was 4 mg / ml.

[Example 3]
pNP-β-Glc degradation activity The degradation activity of pNP-β-Glc was examined. 10 μL of the crude enzyme solution, 50 μL of 0.2 M sodium phosphate buffer (pH 7.0), 50 μL of 20 mM pNP-β-Glc aqueous solution and water were added to make a total of 200 μL, followed by reaction at 37 ° C. Since the BGL11-1 crude enzyme solution had high activity, the crude enzyme solution diluted 100-fold with 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS was used. The absorbance change at 405 nm per minute (Δ405) based on p-nitrophenol (pNP) liberated by hydrolysis of pNP-β-Glc was 0.244 for the BGL11-1 crude enzyme solution, and C-1 crude enzyme The liquid was 0.000.
From the above, it was suggested that AOBGL11p has β-glucosidase activity.

Using pNP-β-Glc as a substrate, the optimum temperature, optimum pH, thermal stability and pH stability of AOBGL11p were examined (FIG. 2 (FIGS. 2A to 2D)).
The BGL11-1 crude enzyme solution was diluted 5000 times with buffer A (protein concentration 1.3 μg / ml).

Optimal temperature:
The reaction solution was 20 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2 M sodium phosphate buffer (pH 6.5), 20 mM pNP-β-Glc, and water to make a total of 400 μl, 15 minutes after the start of the reaction 100 μl was sampled at 30 minutes and 45 minutes and mixed with 100 μl 0.2 M sodium carbonate solution, and the absorbance at 405 nm was measured to obtain Δ405. FIG. 2A shows the ratio of Δ405 when the reaction is performed at each temperature with 45 ° C. at which Δ405 is maximized as 1. As a result, it was found that 45-50 ° C. was the optimum temperature for the reaction.

Optimum pH:
The reaction solution was adjusted to a total of 400 μl by adding 20 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2M buffer, 20 mM pNP-β-Glc, and water. As the buffer, sodium acetate buffer was used at pH 4.0-6.0, and sodium phosphate buffer was used at pH 6.0-8.0. Sampling and measurement were performed in the same manner as described above, and the ratio of Δ405 when reacted at each pH with respect to the maximum value of Δ405 is shown in FIG. 2B. As a result, it was found that pH 6.0-7.0 was the optimum reaction pH.

Thermal stability:
The crude enzyme solution (1.3 μg / ml) diluted 5000 times was kept at 30 ° C., 37 ° C., 45 ° C. and 50 ° C. for 10 minutes, and then cooled with ice. The reaction solution was 5 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2 M sodium phosphate buffer (pH 6.5), 20 mM pNP-β-Glc, and water to make a total of 100 μl, and at 37 ° C. for 45 minutes. After the reaction, 100 μl of 0.2 M sodium carbonate solution was added and the absorbance at 405 nm was measured. The ratio when treated at each temperature to the absorbance at 405 nm after 45 minutes with the enzyme solution that was not heat-treated was determined and is shown in FIG. 2C. AOBGL11p was stable up to 37 ° C. when treated for 10 minutes, but was deactivated to about half when treated at 45 ° C., and the activity was almost lost when treated at 50 ° C.

pH stability:
The crude enzyme solution was added at pH 4.5, 5.0, 5.5, 6.0 (0.2 M acetate buffer), pH 6.0, 6.5, 7.0, 7.5, 8.0 (0.0. 2M sodium phosphate buffer) was diluted 5000 times with each buffer, kept at 37 ° C. for 1 hour, and then ice-cooled. The reaction solution was 5 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2 M sodium phosphate buffer (pH 6.5), 20 mM pNP-β-Glc, and water to make a total of 100 μl, and at 37 ° C. for 45 minutes. After the reaction, 100 μl of 0.2 M sodium carbonate solution was added and the absorbance at 405 nm was measured. The ratio of the absorbance when held at each pH to the absorbance when held at pH 6.5, which had the highest activity, was determined and is shown in FIG. 2D. AOBGL11p was found to be most stable around pH 6.5.

[Example 4]
Cloning of AOBGL11 cDNA The BGL11-1 strain was cultured in 10 ml of enzyme production medium, and the cells were collected by filtration. The cells were frozen with liquid nitrogen, ground in a mortar, and then total RNA was extracted with RNeasy (QIAGEN). CDNA was synthesized by SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies). Using this as a template, PCR was performed with KOD-plus (Toyobo) using primers AOBGL11-F and AOBGL11-R. The obtained DNA fragment of about 2.52 kbp was cloned by using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) to obtain the cDNA of AOBGL11 to obtain plasmid pCR-AOBGL11 cDNA. When the base sequence was confirmed, it was as shown in SEQ ID NO: 1. FIG. 3 shows a comparison between the genomic DNA sequence of AOBGL11 and the cDNA sequence.

[Example 5]
Production of AOBGL11p in Yeast Construction of Yeast Expression Vector and Transformation of Yeast About 2.52 kbp of DNA fragment obtained by digesting plasmid pCR-AOBGL11 cDNA with EcoRI was transformed into yeast expression vector pYE22m (Biosci. Biotech. Biochem , 59, 1221-1228, 1995), and the one in which AOBGL11 was inserted in the direction of expression from the GAPDH promoter of the vector pYE22m was selected and designated pYE-AOBGL3c. S. cerevisiae EH13-15 strain (trp1, MATα) (Appl. Microbiol. Biotechnol., 30, 515-520, 1989) was used as a parent strain for transformation.
EH13-15 strain was transformed by lithium acetate method using plasmids pYE22m (control) and pYE-AOBGL11 (for AOBGL11 expression), respectively. The transformed strain was SC-Trp (per 1 L, yeast nitrogen base w / o amino acids (DIFCO) 6.7 g, glucose 20 g, and amino acid powder (adenine sulfate 1.25 g, arginine 0.6 g, aspartic acid 3 g, Glutamic acid 3 g, histidine 0.6 g, leucine 1.8 g, lysine 0.9 g, methionine 0.6 g, phenylalanine 1.5 g, serine 11.25 g, tyrosine 0.9 g, valine 4.5 g, threonine 6 g, uracil 0.6 g 1) containing 1.3 g) and those growing on an agar medium (2% agar) were selected.

The strain obtained by transformation with plasmid pYE22m was designated as CY strain, and the strain obtained by transformation with plasmid pYE-AOBGL11 was designated as AOBGL11-Y strain.
The selected CY strain and AOBGL11-Y strain were inoculated with 1 platinum ear in 10 mL of SC-Trp liquid medium supplemented with 1/10 volume of 1M potassium phosphate buffer, and cultured with shaking at 30 ° C. and 125 rpm for 2 days. . The obtained culture was separated into a culture supernatant and cells by centrifugation. The culture supernatant was concentrated to ultrafiltration with Amicon (registered trademark) Ultra-15 50k (Merck), buffer-substituted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS, and about 1 ml of culture was obtained. A supernatant concentrate was obtained.
The cells are suspended in 1 ml of 50 mM sodium phosphate buffer (pH 7.0) and 0.1% CHAPS solution. The cells are disrupted with glass beads, and the supernatant obtained by centrifugation is disrupted. Liquid.
Take 20 μl of the culture supernatant concentrate or cell disruption solution, add 1 μl of 2% X-β-Glc / DMF solution and react at room temperature for 5 minutes. Only the cell disruption solution derived from AOBGL11-Y strain is blue. And was suggested to have X-β-Glc activity.

Measurement of pNP-β-Glc activity The degradation activity of pNP-β-Glc was examined. 10 μL of the crude enzyme solution, 50 μL of 0.2 M sodium phosphate buffer (pH 7.0), 50 μL of 20 mM pNP-β-Glc aqueous solution and water were added to make a total of 200 μL, followed by reaction at 37 ° C. Since the BGL11-1 crude enzyme solution had high activity, the crude enzyme solution diluted 100-fold with 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS was used. The absorbance change at 405 nm per minute (Δ405) based on p-nitrophenol (pNP) released by hydrolysis of pNP-β-Glc was 0.068 in the AOBGL11-Y crude enzyme solution, and CY crude enzyme. The liquid was 0.000.

[Example 6]
Mogrol glycoside hydrolysis activity of AOBGL11p produced by Aspergillus mogroside V was used as a substrate. Mogroside V was 50 μg / mL, 50 mM sodium phosphate buffer (pH 7.0), the above BGL11-1 crude enzyme solution or its diluted solution (20 μL) to a total volume of 100 μL, and reacted at 37 ° C. for 1 hour. As a control, the above C-1 crude enzyme solution was reacted in the same manner. The reaction solution was subjected to SepPakC18 500 mg (Waters) washed with methanol and equilibrated with water. After washing with 40% methanol, elution was performed with 80% methanol, followed by drying at a speed bag. Dissolved in 100 μL of water and subjected to HPLC.

The analysis conditions of HPLC are as follows.
Column: Cosmo Seal 5C ₁₈ -AR-II 4.6 mmI. D. x250mm (Nacalai Tesque)
Mobile phase: A; acetonitrile, B; water B conc. 90% → 30% 60 minutes linear gradient
Flow rate: 1 ml / min Temperature: 40 ° C
Detection: UV 203nm

The results are shown in FIG.

When reacted with a 100-fold diluted BGL11-1 crude enzyme solution, a portion of the substrate mogroside V is hydrolyzed to produce a tetrasaccharide mogrol glycoside. From the retention time, this tetrasaccharide mogrol glycoside was considered to be siamenoside I in which the β-1,6 bond of gentiobiose added to the 3-position of mogrol V was hydrolyzed.
In the case of reacting with an enzyme solution diluted 10 times, hydrolysis further progressed, and tetrasaccharide, trisaccharide (mogroside IIIE from the retention time) and disaccharide mogrol glycoside were produced.
Further, when the enzyme solution was reacted without diluting, no glycoside including mogroside V added as a substrate was detected, and mogrol which was an aglycon was detected.
That is, it has been clarified that the hydrolysis reaction of mogroside V by the BGL11-1 crude enzyme solution proceeds in such a manner that glucose is cleaved one by one and finally hydrolyzed to aglycone, mogrol. Moreover, it is shown that the progress of hydrolysis of the mogrol glycoside can be controlled by adjusting the concentration of the enzyme solution. Further, the result of the reaction between the C-1 crude enzyme solution and mogroside V is shown in FIG. 5, and this result shows that the C-1 crude enzyme solution does not advance hydrolysis of mogroside V.
[Example 7]

Hydrolysis activity of mogrol glycosides by AOBGL11p produced in yeast The hydrolysis activity of mogrol glycosides in the cell disruption liquids of CY and AOBGL11-Y strains was tested.
Substrate 50 μg / ml, enzyme solution 20 μl, 50 mM sodium phosphate buffer (pH 6.0) were mixed to make a total volume of 100 μl, and reacted at 37 ° C. for 1 hour. The reaction solution was purified and subjected to HPLC analysis. The purification method of the reaction solution and the HPLC conditions are as described above.

When the reaction was carried out with a cell disruption solution derived from the CY strain, the product could not be detected with any of the mogrol glycosides. On the other hand, when mogroside V is used as a substrate in the cell disruption solution derived from the AOBGL11-Y strain, siamenoside I, mogroside IIIE, mogrol disaccharide glycoside, and mogrol are produced as products. Thus, it was found that AOBGL11 shows the same activity when expressed in yeast as in the case of expressing it in Aspergillus.

The present invention provides a method for producing mogrol and / or mogrol glycoside by hydrolyzing mogroside V using AOBGL11p which is a glucoside hydrolase derived from Aspergillus.

Claims (15)

1. A step of reacting a protein selected from the group consisting of the following (a) to (c) with a mogrol glycoside to hydrolyze at least one glucoside bond of the mogrol glycoside, and / or A method for producing mogrol glycoside.
   (A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
   (B) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted and / or added, and hydrolyzes at least one glucoside bond of the mogrol glycoside. A protein having the activity of:
   (C) a protein having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside
2. The production method according to claim 1, wherein the mogrol glycoside to be reacted with protein is mogroside V, mogroside IV, siamenoside I, 11-oxomogroside, mogroside includes mogroside I, mogroside IVA, mogroside III, mogroside IIIA ₁ , mogroside IIIA _2. The method according to claim 1, which is at least one selected from the group consisting of Mogroside IIIE, Mogroside IIA, Mogroside IIA ₁ , Mogroside IIA ₂ , Mogroside IIB, Mogroside IIE, Mogroside IA ₁ , Mogroside IE ₁ .
3. The method according to claim 1 or 2, wherein the mogrol glycoside is at least one selected from the group consisting of mogroside V, mogroside IIIE, and siamenoside I.
4. The method according to any one of claims 1 to 3, wherein the mogrol glycoside is mogroside V.
5. The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a glucoside bond between glucose added at the 3-position of mogrol and mogrol which is an aglycone, and at the 24-position of mogrol. Β1,6-glucoside bond of added branched trisaccharide, β-1,6-glucoside bond of gentiobiose added to position 24 of mogrol, β-1,2-glucoside bond of sophorose added to position 24 of mogrol, The method according to any one of claims 1 to 4, which is any one of glucoside bonds between glucose added to position 24 of mogrol and mogrol which is an aglycone.
6. An enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to (e) is introduced into a host cell is contacted with the mogrol glycoside, and the mogrol A method for producing mogrol and / or mogrol glycoside, comprising hydrolyzing at least one glucoside bond of the glycoside.
   (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
   (B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
   (C) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added, and at least one glucoside bond of the mogrol glycoside is hydrolyzed. A polynucleotide encoding a protein having an activity of degrading;
   (D) encoding a protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. A polynucleotide to:
   (E) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and at least one of the mogrol glycosides Polynucleotide encoding protein having activity of hydrolyzing glucoside bond
7. The method according to claim 6, wherein the polynucleotide is inserted into an expression vector.
8. The method according to claim 6 or 7, wherein the transformed cell is a transformed gonococcus, a transformed yeast, a transformed bacterium, or a transformed plant.
9. Moguroru glycoside contacting said enzyme agent, mogroside V, mogroside IV, siamenoside I, 11- Okisomoguroshido, the mogroside, mogroside I, mogroside IVA, mogroside III, mogroside IIIA _1, mogroside IIIA _2, mogroside IIIE, mogroside The method according to any one of claims 6 to 8, which is at least one selected from the group consisting of IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIB, mogroside IIE, mogroside IA ₁ , mogroside IE ₁ .
10. The method according to any one of claims 6 to 9, wherein the mogrol glycoside is at least one selected from the group consisting of mogroside V, mogroside IIIE, and siamenoside I.
11. The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a glucoside bond between glucose added at the 3-position of mogrol and mogrol which is an aglycone, and at the 24-position of mogrol. Β1,6-glucoside bond of added branched trisaccharide, β-1,6-glucoside bond of gentiobiose added to position 24 of mogrol, β-1,2-glucoside bond of sophorose added to position 24 of mogrol, The method according to any one of claims 6 to 10, which is any one of glucoside bonds between glucose added at position 24 and / or mogrol which is an aglycone.
12. A method for producing mogrol and / or mogrol glycoside, comprising culturing a non-human transformant into which a polynucleotide selected from the group consisting of the following (a) to (e) is introduced.
    (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
    (B) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
    (C) The amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added, and at least one glucoside bond of the mogrol glycoside is hydrolyzed. A polynucleotide encoding a protein having an activity of degrading;
    (D) encoding a protein having an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of the mogrol glycoside. A polynucleotide to:
    (E) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and at least one of the mogrol glycosides Polynucleotide encoding protein having activity of hydrolyzing glucoside bond
13. The method according to claim 10, wherein the polynucleotide is inserted into an expression vector.
14. The method according to claim 12 or 13, wherein the transformant is a transformed gonococcus, a transformed yeast, a transformed bacterium, or a transformed plant.
15. The at least one glucoside bond is a β-1,6-glucoside bond of gentiobiose added at the 3-position of mogrol, a glucoside bond between glucose added at the 3-position of mogrol and mogrol which is an aglycone, at the 24-position of mogrol Β-1,6-glucoside bond of added branched trisaccharide, β-1,6-glucoside bond of gentiobiose added at position 24 of mogrol, β-1,2-glucoside of sophorose added at position 24 of mogrol The method according to any one of claims 12 to 14, which is either a bond and / or a glucoside bond between glucose added to position 24 of mogrol and mogrol which is an aglycon.
    

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